Non-virulent Porphyromonas gingivalis mutant

ABSTRACT

A non-virulent, recA defective mutant of Porphyromonas gingivalis. A pharmaceutical composition comprising the Porphyromonas gingivalis strain of the present invention.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present Application is a continuation of U.S. patentapplication Ser. No. 09/762,618, titled “Nonvirulent Strain ofPorphyromonas Gingivalis” and filed Feb. 9, 2001, now U.S. Pat. No.6,585,977 issued Jul. 1, 2003; which is a national phase filing under 35U.S.C. §371 of PCT Application No. PCT/US99/18197, titled “Non-virulentPorphyromonas Gingivalis Mutant” and filed Aug. 11, 1999; which is acontinuation of U.S. patent application Ser. No. 09/133,089, titled“Non-Virulent Porphyromonas Gingivalis Mutant” and filed Aug. 12, 1998,now U.S. Pat. No. 6,254,863, issued Jul. 3, 2001; and the presentApplication is a continuation of U.S. patent application Ser. No.09/803,766, titled “Nonvirulent Strain of P. Gingivalis” and filed Mar.12, 2001, now U.S. Pat. No. 6,586,227 issued Jul. 1, 2003; the contentsof which are incorporated in this disclosure by reference in theirentirety.

BACKGROUND

[0002] Periodontitis is an inflammatory disease of the tissuessurrounding the teeth characterized by loss of the periodontal ligamentattachment and alveolar bone support of the tooth. Periodontitis affectsmore than 49 million people in the United States and hundreds ofmillions of people worldwide and has been reported as a risk factor forcardiovascular disease and pre-term delivery of low-birth-weightinfants. The most common cause of periodontitis is chronic Gram-negativebacterial infections. Among the Gram-negative bacteria implicated as acause of periodontitis, Porphyromonas gingivalis is the major componentof the flora in over 90% of adult periodontitis lesions.

[0003] Besides being a major etiological agent in adult humanperiodontitis, Porphyromonas gingivalis also causes aspiration pneumoniaand necrotizing pneumonia, abscesses in brain, genitourinary tract andlung, as well as mediastinitis. By contrast, P. gingivalis is notnormally found at healthy sites nor is it found in patients withgingivitis but with no accompanying periodontitis.

[0004] The current therapy for periodontitis is directed towardidentifying, removing and controlling the etiologic factors, and thencorrecting the defects these pathogens have caused. These therapiesinclude scaling and root planing, chemotherapy, periodontal surgery andperiodic maintenance therapy. However, these treatments are not entirelyeffective because, for example, the pathogens can become resistant tochemotherapeutic agents.

[0005] Several potential virulence factors have been identified whichappear to relate to the pathogenicity of P. gingivalis in periodontitis.These factors include fimbriae (adhesins), capsule (antiphagocytosis),lipopolysaccharide (bone resorption), proteases (specific andgeneralized tissue destruction) and a variety of toxic by-products(e.g., ammonia). Some of these factors have been purified andbiochemically characterized. However, the specific roles, interactions,relative importance and regulation of these factors remain to bedetermined.

[0006] Therefore, there remains a need for effective prevention andtreatment for periodontitis. Further, there remains a need for amodified strain of P. gingivalis that can be used as a host geneticbackground to determine the specific roles, interactions, relativeimportance and regulation of the potential virulence factors produced bywild-type P. gingivalis.

SUMMARY OF THE INVENTION

[0007] According to one embodiment of the present invention, there isprovided a non-virulent, recA defective mutant of Porphyromonasgingivalis. According to another embodiment of the present invention,there is provided a Porphyromonas gingivalis strain which is depositedat ATCC under accession number 202109.

[0008] According to another embodiment of the present invention, thereis provided a pharmaceutical preparation comprising a mutant ofPorphyromonas gingivalis according to the present invention.

[0009] According to another embodiment of the present invention, thereis provided a method of decreasing the growth rate or reproduction rateof Porphyromonas gingivalis in a mammal, such as a human. The methodcomprises the step of administering to the mammal at least one dose of anon-virulent, recA defective mutant of Porphyromonas gingivalis, such asat least one dose of a Porphyromonas gingivalis strain which isdeposited at ATCC under accession number 202109.

[0010] According to another embodiment of the present invention, thereis provided a method of preventing or treating a Porphyromonasgingivalis infection such as periodontitis in a mammal, such as a human.The method comprises the step of administering to the mammal at leastone dose of Porphyromonas gingivalis according to the present invention.

[0011] The methods of the present invention can be performed byadministering to the mutant with the at least one dose of anon-virulent, recA defective mutant of Porphyromonas gingivalis via aroute selected from the group consisting of a subcutaneous route, anintravenous route and an intramuscular route, among other routes. In apreferred embodiment, the methods of the present invention includeadministering at least one dose of a non-virulent, recA defective mutantof Porphyromonas gingivalis, wherein the dose is between about a 1×10³and 1×10⁷ bacteria per kg of body weight of the mammal. More preferably,the dose is between about 1×10⁵ and 1×10⁶ bacteria per kg of body weightof the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures where:

[0013]FIGS. 1 and 2 show the results of Southern blot analyses ofallelic exchange mutants of P. gingivalis to confirm the presence of theermF-ermAM cassette in a predicted location;

[0014]FIG. 3 is a bar graph showing the results of an assay for argininespecific proteolytic activity of P. gingivalis FLL32, FLL33 and W83 inthe presence or absence of L-cysteine;

[0015]FIGS. 4 and 5 are bar graphs showing the results of an assay forthe localization of arginine-specific proteolytic activity and forlysine-specific proteolytic activity, respectively, of P. gingivalisFLL32, FLL33 and W83 in the presence or absence of L-cysteine;

[0016]FIGS. 6 and 7 show the results of Northern blot analyses of prpRIand prtP protease genes, respectively, of P. gingivalis FLL32, FLL33 andW83;

[0017]FIG. 8 shows the results of an analysis by SDS-PAGE of the abilityof P. gingivalis FLL32, FLL33 and W83 to degrade purified C3 complementprotein; and

[0018]FIG. 9 is a graph showing the results of accumulation of C3fragments on the bacterial surface of FLL32, FLL33 and W83.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention involves the discovery of a non-virulentmutant of Porphyromonas gingivalis. This mutant, designated FLL32, hasbeen found to convey protection against the wild-type Porphyromonasgingivalis W83 in mammals when the mammal was immunized with the mutantstrain FLL32. Further, FLL32 can be used as a host genetic background todetermine the specific roles, interactions, relative importance andregulation of the potential virulence factors produced by wild-type P.gingivalis.

[0020] All publications mentioned in this document are incorporated byreference in their entirety.

[0021] A deposit of Porphyromonas gingivalis mutant strain FLL32 hasbeen made at the ATCC depository, 10801 University Blvd., Manassas, Va.,US 20110-2209 on Apr. 8, 1998, under the accession number 202109. Thisdeposit shall be viably maintained, replacing it if it becomesnon-viable, for a period of 30 years from the date of the deposit, orfor 5 years from the last date of request for a sample of the deposit,whichever is longer, and made available to the public withoutrestriction in accordance with the provisions of the law. TheCommissioner of Patents and Trademarks, upon request, shall have accessto the deposit.

[0022] In summary, the FLL32 strain was isolated during construction ofa mutant recA⁻ mutant of P. gingivalis W83 wild-type by allelic exchangemutagenesis. The FLL32 strain was recA⁻ and lacked black pigmentationand β-hemolytic activity on blood agar. Further, the FLL32 strain wasdeficient in proteolytic activity and significantly more sensitive to UVirradiation than the wild-type W83 strain.

[0023] The FLL32 strain exhibited substantially reduced virulence whenintroduced into mammals and protected those animals immunized with thatstrain against subsequent infection by the wild-type strain W83.Further, in Western blot experiments of whole cell extracts, uniqueimmunoreactive bands were found in FLL32 using sera from immunizedanimals. The FLL32 strain is the first nonvirulent recA⁻ strain of P.gingivalis shown to protect mammals against subsequent infection by thewild-type P. gingivalis.

Isolation and Characterization of P. gingivalis FLL32 Strain

[0024] Porphyromonas gingivalis FLL32 was isolated and characterized asfollows. First, the recA homolog gene was cloned from wild-type W83.Next, the recA homolog gene was sequenced. Then, an isogenic recA⁻mutant of P. gingivalis W83 designated FLL32 was constructed by allelicexchange mutagenesis and the presence of the defective recA DNA in theP. gingivalis FLL32 strain confirmed by Southern blot analyses.

[0025] Next, the phenotype and UV sensitivity of P. gingivalis FLL32strain was determined, as well as its arginine and lysine specificproteolytic activity. Additionally, the amount of its mRNA transcriptfor the major protease genes was determined. Then, the amount of its C3complement protein degradation was determined and the amount of C3accumulation on the surface of the P. gingivalis FLL32 strain wasdetermined. Finally, the virulence of the P. gingivalis FLL32 strain andprotective effect of immunization by the FLL32 strain against subsequentchallenge by the wild-type was examined.

[0026] (a) Cloning of the recA Homolog Gene from P. gingivalis W83

[0027] The recA homolog gene was cloned from P. gingivalis W83 asfollows. First, degenerate oligonucleotide primers (Dybvig, K., et al.,“Degenerate oligonucleotide primers for enzymatic amplification of recAsequences from gram-positive bacteria and mycoplasmas.” J. Bacteriol.174, 2729-2732, 1992) were used in a polymerase chain reaction (PCR) toamplify a 320 bp fragment of the recA sequence of P. gingivalis W83.This PCR fragment was ³²P labeled and used to screen a λ DASHrecombinant phage bank P. gingivalis W83 for the presence of hybridizingclones. Ten of 1×10 ³ phage clone plaques (1.0%) hybridized with theprobe.

[0028] The hybridizing phage plaques were then amplified and absorbedonto maltose-grown E. coli cells. DNA from the phage clones was isolatedusing the Promega Lambda Wizard DNA Purification system (Promega,Corporation, Madison, Wis.). NotI-BamHI cleavage of purified DNA fromtwo of the recombinants, designated L2 and L10, revealed that the phageclones had different restriction fragment patterns. L2 contained an 8.0kb and a 6.5 kb fragment that were missing in L10, while L10 containedan 11 kb, a 5.8 kb and a 0.3 kb fragment that were missing in L2. BothL2 and L10 contained a similar 2.1 kb fragment. These data indicatedthat the L2 and L10 clones were independent clones and not siblings froma single cloning event.

[0029] The L10 clone was chosen for further study because it had thesmaller fragment insert. Southern blot hybridization using the³²P-labeled 0.3 kb PCR fragment of the recA gene from the chromosome ofW83 was used as a probe to identify the hybridizing fragment. Theplasmid pUC19 was used to subclone a 2.1 kb hybridizing BamHI fragmentfrom L10. This clone was designated pFLL26.

[0030] (b) Nucleotide Sequencing of the recA Homolog Gene

[0031] Both strands of the 2.1 kb hybridizing BamHI fragment from theL10 clone carried on pFLL26 were sequenced and one 1.02 kb open readingframe corresponding to a 36 kDa protein was detected, GenBank AccessionNumber U70054 (Fletcher et al., 1997). There was a start codon at baseposition 774. A purine-rich sequence found in E. coli ribosome bindingsites was also seen three bases upstream from the initiation site.Sequences resembling procaryotic-10 and-35 promoter regions weredetected at base positions 749 and 729 respectively. The calculated G+Cratio for the recA homolog gene was 50% which is close to the ratio of46 to 48% previously reported for genomic P. gingivalis DNA (Shah andCollins, 1988).

[0032] A comparison of the amino acid sequence of this gene with theNational Center for Biotechnology Information genetic sequence databankrevealed a similarity of approximately 90, 86 and 82 percent to the RecAproteins from Bacteroides fragilis, Prevotella ruminicola, GenBankAccession Number U21227, and Mycobacterium smegmatis, GenBank AccessionNumber X99208, respectively. (Goodman and Woods, Molecular Analysis ofthe Bacteroides fragilis recA Gene, Gene 94, pp. 77-82, 1990) Further,regions between amino acids 68 to 81 and 266 to 288 revealed conservedATP binding domains.

[0033] (c) Construction of a recA⁻Mutant in P. gingivalis W83

[0034] An isogenic recA⁻ mutant of P. gingivalis W83 was constructed byallelic exchange mutagenesis as follows. The nucleotide sequence of thecloned recA fragment revealed a unique HincII restriction site at bp 435of the open reading frame (Fletcher, H. M. et al., “Nucleotide sequenceof Porphyromonas gingivalis W83 recA homolog and construction of arecA-deficient mutant.” Infect. Immun. 65, 4592-4597, 1997). To utilizethis site, a 1.8 kb EcoRI-PstI fragment containing the intact recA genewas subcloned into EcoRI-PstI cleaved pUC19. The resulting plasmid,pFLL23, was digested with HincII and ligated with the 2.1 kb ermF-ermAMcassette from pVA2298 to produce recombinant plasmid designated pFLL24.(See Fletcher, H. M., Schenkein, H. A., Morgan, R. M., Bailey, K. A.,Berry, C. R., and Macrina, F. L. (1995). Virulence of a mutant ofPorphyromonas gingivalis W83 that is defective in the prtH gene. Infect.Immun. 63, 1521-1528).

[0035] Then, the recombinant plasmid pFLL24 was used as donor DNA inelectroporation of P. gingivalis W83. Since the pFLL24 plasmid wasunable to replicate in P. gingivalis, Clindamycin resistant (Cc^(r))transformants could only arise as a result of an integration into thewild-type gene on the chromosome. Two double crossover events werepredicted between the regions flanking the erm marker and the wild-typerecA gene on the chromosome that would result in a replacement of asegment of the wild-type gene with a fragment conferring clindamycinresistance.

[0036] Following electroporation and plating on selective medium, 15Cc^(r) colonies were detected after a 7 day incubation period. Thesecolonies were replica plated onto selective medium and exposed to UVlight to determine their sensitivity. Four UV sensitive colonies,designated FLL32, FLL33, FLL34 and FLL35, were chosen from the unexposedplate for further study.

[0037] To confirm the presence of the ermF-ermAM cassette in thepredicted location, that is interrupting the recA DNA, Southern blotanalyses were performed on the total cellular DNA from P. gingivaliswild-type W83, as a control, and from the Cc^(r) transformants FLL32,FLL33, FLL34 and FLL35. Their DNA was cleaved with BamHI,electrophoresed through 0.7% agarose, bidirectionally transferred tonitrocellulose and probed with ³²P labeled pFLL23 and pVA2198. If theDNA was digested with BamHI, a predicted 2.1 kb fragment would be seen.If the DNA was not digested with BamHI, a predicted 4.2 kb fragmentwould be seen. Since the ermF-ermAM cassette is missing a BamHI site,any of the four Cc^(r) transformants with the ermF-ermAM cassetteinterrupting the recA DNA sequence should have shown a 4.2 kb fragmentbut not a 2.1 kb fragment.

[0038] Referring now to FIGS. 1 and 2, there are shown the results ofthe Southern blot analyses of allelic exchange mutants of P. gingivalisto confirm the presence of the ermF-ermAM cassette in the predictedlocation. As can be seen in FIG. 1, the predicted 2.1 kb fragment wasseen in the wild-type P. gingivalis W83, lane A, using the ³²P-labeledpFLL23 that carries the P. gingivalis recA homolog as a probe, indicatedthe presence of the recA DNA. In contrast, a 4.2 kb fragment was presentin each of the four Cc^(r) mutants of W83, lanes B-E, FLL32, FLL33,FLL34 and FLL35, respectively, and indicated the presence of the recADNA sequence interrupted by the ermF-ermAM cassette.

[0039] As can be seen in FIG. 2, using pVA2198, which carried theermF-ermAM cassette as a probe, revealed an identical 4.2 kb hybridizingfragment present in the four Cc^(r) mutants, lanes B-E, FLL32, FLL33,FLL34 and FLL35, respectively, but not in the wild-type W83, lane A,indicating the presence of the ermF-ermAM cassette only in thetransformants. pUC19 vector sequences used as a control did nothybridize with W83 or any of the four Cc^(r) mutants, FLL32, FLL33,FLL34 and FLL35 (data not shown). These data indicated thatrecombination had occurred in the four Cc^(r) mutants, FLL32, FLL33,FLL34 and FLL35, resulting in the wild-type recA gene being interruptedby the ermF-ermAM cassette in FLL32, FLL33, FLL34 and FLL35.

[0040] (d) Phenotypic Characterization of P. Gingivalis W83 recA Mutants

[0041] The phenotype of P. gingivalis W83 recA mutants were initiallycharacterized as follows. The recA mutants, FLL32, FLL33, FLL34 andFLL35, were plated on Brucella blood agar plates (Anaerobic Systems,Inc., San Jose, Calif.) to determine if any pleotropic effects wereassociated with inactivation of the recA gene. Two classes of mutantswere observed. The first class, a single colony FLL32, was unpigmentedand displayed significantly less β-hemolysis than the wild-type W83. Thesecond class contained three strains, FLL33, FLL34 and FLL35, all ofwhich displayed similar β-hemolytic activity and black pigmentation asthe wild-type W83. FLL32 and FLL33 were chosen for further study asrepresentatives of their respective classes. A generation time of 3hours was determined for W83 and of 3.5-4 hours for both FLL32 andFLL33.

[0042] (e) Determination of the UV Sensitivity of P. Gingivalis W83 recAMutants

[0043] To confirm the loss of activity of the P. gingivalis RecAprotein, the relative sensitivity of the wild-type and recA⁻ strains toUV irradiation was determined as follows. Wild-type W83 and recA⁻mutants FLL32 and FLL33 were exposed to 1000 μjoules of UV irradiation.There was an 80% survival of the wild-type W83 strain in contrast to the18% survival for FLL32 and FLL33. When wild-type W83 and mutants FLL32and FLL33 were exposed to 2000 μjoules of UV irradiation, there was 40%survival of the wild-type W83 cells compared to 0% survival for therecA⁻ mutants FLL32 and FLL33. These data indicated that the recA geneof P. gingivalis W83 plays an important role in repairing DNA damagecaused by UV irradiation and that both FLL32 and FLL33 were recAdefective.

[0044] (f) Determination of the Arginine and Lysine Specific ProteolyticActivity of FLL32, FLL33 and W83

[0045] The arginine specific proteolytic activity of P. gingivalis W83recA mutants was determined by assaying whole cell preparations fromeach of the three strains of P. gingivalis, FLL32, FLL33 and W83 forproteolytic activity using N-α-benzoyl-DL-arginine p-nitroanilide(BAPNA). Each strain of P. gingivalis was grown for 48 hours to late logphase (OD₆₀₀ of 1.2) in 500 ml BHI broth supplemented with hemin andvitamin K. The cells were then washed in PBS (pH 7.3) and resuspended toan OD₆₀₀ of 0.3. 50 μl of the cell samples were incubated for 10 min at37° C. in 50 mM Tris-HCl (pH 7.0), and 1 mMα-N-benzoyl-arginine-DL-p-nitroanilide (BAPNA) in the presence orabsence of 0.5 mM L-cysteine. The control contained buffer alone.Hydrolysis of BAPNA was monitored by the change of absorbance at 410 nm.

[0046] Referring now to FIG. 3, there is shown a bar graph of the assayresults. As can be seen, the wild-type W83 (−Cys) showed moreproteolytic activity than FLL33 (−Cys), while FLL32 (−Cys) did not showsignificantly more proteolytic activity than the control. The activityfrom all three strains was enhanced in the presence of cysteine (+Cys)but the relative rates of proteolytic activity remained the same. Thereduction of proteolytic activity seen in FLL33 compared to W83 could berelated to the longer generation time for the recA⁻ strains compared tothe wild-type W83.

[0047] Localization of the arginine-specific proteolytic activity andfor lysine-specific proteolytic activity in the recA⁻ strains wasdetermined as follows. First, extracellular proteolytic activity wastested. Ammonium sulfate was added to 500 ml of culture supernatant fromcells grown to late log phase (OD₆₀₀ of 1.2) to 100% saturation. Theprecipitate was resuspended in 3 ml of PBS (pH 7.3), dialyzed againstthe same buffer, and then stored at −20° C.

[0048] Referring now to FIGS. 4 and 5, there are shown bar graphsshowing the results of an assay for the localization ofarginine-specific proteolytic activity and for lysine-specificproteolytic activity. As can be seen, the FLL33 showed moreextracellular arginine-specific proteolytic activity and morelysine-specific proteolytic activity than the wild-type W83 (−Cys).FLL32 did not show significantly more arginine-specific proteolyticactivity or lysine-specific proteolytic activity than the control.Further, the extracellular arginine-specific proteolytic activity ofboth W83 and FLL33, but not FLL32, was enhanced in the presence ofcysteine (+Cys).

[0049] Next, intracellular proteolytic activity was tested. The cellsfrom the above experiment were washed in PBS (pH 7.4), and thenresuspended in the same buffer to a final volume of 10 ml. 1 ml aliquotswere transferred to microcentrifuge tubes containing 0.5 volume of 0.1mm zirconium beads (Biospec Products, Inc. Bartlesville, Okla.), thenlysed in a Mini-Bead Beater homogenizer (Biospec Products) for 3 min.Beads and cellular debris were removed by centrifugation at 12,000×g for5 min to obtain a clear lysate. Using 100 μg of protein per assay,similar intracellular arginine- and lysine-specific proteolyticactivities were observed for the W83 and FLL33 strains, but there was nosignificant intracellular arginine- or lysine-specific proteolyticactivities for FLL32 (data not shown).

[0050] (g) Comparison of the Presence and Amount of mRNA Transcript forthe Major Protease Genes in FLL32, FLL33 and W83

[0051] The loss of proteolytic activity in strain FLL32 could haveresulted either from a lack of transcription or translation of the gene,or from a lack of post-translational activation of the precursorproduct. In order to determine the cause of the loss of proteolyticactivity in FLL32, the presence and amount of mRNA transcript for themajor protease genes in FLL32, FLL33 and W83 was determined as follows.

[0052] First, total RNA was isolated using the Qiagen RNeasy Kit(Qiagen, Valencia, Calif.) from the wild-type W83 strain and from theFLL32 and FLL33 mutants grown to mid-log phase (OD₆₀₀ of 0.2). Uniqueoligonucleotide primers for prtP (as disclosed in Barkocy-Gallagher, G.A. et al., “Analysis of the prtP gene encoding porphypain, a cysteineproteinase of Porphyromonas gingivalis.” J. Bacteriol. 178, 2734-2741,1996), prpRI (Aduse Opoku, J. et al., “Characterization, geneticanalysis, and expression of a protease antigen (PrpRI) of Porphyromonasgingivalis W50. ” Infect. Immun. 63, 4744-4754, 1995) and prtRII wereused in RT-PCR to amplify a 1 kb region of the transcripts. Amplifiedproducts of the predicted 1 kb size were observed for all three proteasegene transcripts in all three strains (data not shown). Further, therewere no observed differences seen in the concentration of the amplifiedproduct between the genes of the three strains. Therefore, both FLL32and FLL33 strains produce the same mRNA transcripts for the majorprotease genes in the same amounts as the wild-type W83. As a control,recA intragenic primers amplified the expected 0.72 kb region only inthe wild-type W83 strain.

[0053] The presence of the mRNA transcripts for the prpRI and prtPproteases in all three P. gingivalis strains were further confirmed inNorthern blot analysis using an amplified intragenic region of each geneas a probe. Total RNA was extracted from each of the W83, FLL32 andFLL33 strains grown to mid-log phase (OD₆₀₀ of 0.2) using the QiagenRNeasy midi kit (available from Qiagen, Valencia, Calif., according tothe manufacturer's instructions). RNA samples of 1 μg were thenseparated by agarose gel electrophoresis and transferred tonitrocellulose filter according to the method of Sambrook et al.(Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). MolecularCloning: A Laboratory Manual. Second edition. (Cold Spring Harbor, N.Y.:Cold Spring Harbor Laboratory Press)).

[0054] Referring now to FIGS. 6 and 7, there are shown the results ofthe Northern blot analysis of prpRl and prtP protease genes from thethree strains of P. gingivalis W83, FLL33 and FLL32. FIG. 6 shows theresults using a ³²P-labeled specific intragenic region of prpRI as theprobe. FIG. 7 shows the results using a ³²P-labeled specific intragenicregion of prpP as the probe. The size of the transcripts in kb are givenin the left margins. Lane A shows the results for the W83 wild-typestrain, lane B shows the results for the FLL32 mutant strain and lane Cshows the results for the FLL33 mutant strain.

[0055] As can be seen in FIG. 6, the prpRI probe hybridized to 6.3 and4.2 kb transcripts. As can be seen in FIG. 7, the prtP probe hybridizedto 6.6, 4.3 and 3.2 kb transcripts. The 6.3 and 6.6 kb transcripts forthe prpRI and prtP genes, FIGS. 6 and 7 respectively, are consistentwith the known size of those genes transcripts. The presence of thesmaller transcripts could be degraded product or could be transcriptsthat share regions of homology with the protease genes. These resultsconfirm the presence of the mRNA transcripts of the prpRI and prtPprotease genes in all three strains of P. gingivalis, W83, FLL32 andFLL33.

[0056] (h) Determination of the C3 Complement Protein Degradation ofFLL32, FLL33 and W83

[0057] The ability of P. gingivalis FLL32, FLL33 and W83 to degradepurified C3 complement protein was determined as follows. 1 mg/ml of C3was incubated with increasing dilutions of each strain at 37° C. for 30minutes and the supernatant were analysed by SDS-PAGE with 10%separation gels and strained with comassie. The results are shown inFIG. 8 using C3 alone as a control (lane 1), 10⁹ W83 cells/ml (lane 2),5×10⁸ W83 cells/ml (lane 3), 10⁸ W83 cells/ml (lane 4), 10⁹ FLL32cells/ml (lane 5), 5×10⁸ FLL32 cells/ml (lane 6), 10⁸ FLL32 cells/ml(lane 7), 10⁹ FLL33 cells/ml (lane 8), 5×10⁸ FLL33 cells/ml (lane 9) and10⁸ FLL33 cells/ml (lane 10).

[0058] As can be seen, the highest concentration of W83 tested (lane 2)completely degraded the α-chain of C3 with generation of C3b and somelower molecular mass fragments similar to C3c and C3d. The lowestbacterial concentration of W83 tested (lane 4) partially degraded theα-chain, causing both a and α′-chains to be visible. Similar resultswere observed for FLL33 (lanes 8-10). In contrast, the highestconcentration of FLL32 (lane 6) only minimally degraded C3 to C3b andlower molecular mass cleavage fragments. There was no degradation of C3at the lowest bacterial concentration tested for FLL32. Thus, FLL32 isless capable of degrading C3 than either the wild-type W83 or FLL33.

[0059] (i) Assessment of C3 Accumulation on FLL32, FLL33 and W83

[0060] Opsonization of P. gingivalis strains W83, FLL32 and FLL33 wasassessed for the accumulation of C3 fragments on the bacterial surfaceas follows. 5×10⁸ cells/ml of each strain was incubated in pooled humanserum that was diluted 1:3 with Veronal-buffered saline (0.01 M Veronalbuffer, pH 7.5, containing 0.13 M NaCl) and that contained 2 μg/ml¹²⁵I-C3 for 35 minutes. The incubated bacterial samples were then washedand assessed for bound ¹²⁵I-C3 fragments by scintillation counting.Referring now to FIG. 9, it can be seen that W83 failed to accumulatesubstantial amounts of C3 by the end of the incubation period. Incontrast, FLL33 accumulated 3×10⁴ molecules/bacterium and FLL32accumulated 6×10⁴ molecules/bacterium of ¹²⁵I-C3 fragments. Takentogether, these results suggest that FLL32 has an increased capacity tobe opsonized with C3 fragments compared to both W83 and FLL33.

EXAMPLE I Comparison of the Virulence Between FLL32, FLL33 and W83 in aMammal

[0061] A first comparison of the virulence between wild-type W83, mutantstrain FLL32 and mutant strain FLL33 Porphyromonas gingivalis in amammal was made as follows. Sixteen female Balb/c mice (8-10 weeks old,Harlan Sprague Dawley Inc., Indianapolis Ind.) were divided into threegroups, five in Group I, five in Group II and six in Group III. Eachanimal received a single challenge dose of 1×10¹⁰ bacteria P. gingivalisW83 (Group I), FLL33 (Group II) or FLL32 (Group III) by subcutaneous,dorsal surface injection, a dosage of approximately 2×10⁴ bacterial perkg body weight.

[0062] At 24 hours post-challenge, two of the five animals in Group Iand one of the five animals in the Group II had died and the remaininganimals in both Groups I and II appeared cachectic and hunched withruffled hair. Although the animals did not display lesions at the dorsalsurface site of injection (primary site), all had developed spreading,ulcerative abdominal skin lesions (secondary site). All of the remaininganimals in the Group I and three of the four remaining animals in theGroup II died by 48 hours post-challenge. The fifth animal in the GroupII died by the fourth day post-challenge.

[0063] In contrast, all six of the animals in Group III challenged withFLL32 survived the 14 day post-challenge observation period. None of theanimals in Group III had any observable negative effects from thechallenge.

[0064] The data from these challenges were analyzed using Fisher's ExactTest. The analysis found no difference in the virulence between W83 andFLL33(p=1.000). However, the FLL32 strain had a statistical differencein virulence when compared to FLL33 (p=0.002) and W83 (p=0.002).

[0065] A second comparison of the virulence between wild-type W83,mutant strain FLL33 and mutant strain FLL32 Porphyromonas gingivalis ina mammal was made as follows. Seventeen mice Balb/c mice (8-10 weeksold, Harlan Sprague Dawley Inc., Indianapolis Ind.) were divided intothree groups, five in Group IV, six in Group V and six in Group VI. Eachanimal received a single challenge dose of 5×10⁹ bacteria P. gingivalisW83 (Group IV), FLL33 (Group V) or FLL32 (Group VI) by dorsalsubcutaneous surface injection.

[0066] At 24 hours post-challenge, one of five animals in Group IV haddied and the remaining four had developed ulcerated abdominal skinlesions. By 48 hours post-challenge, three of the remaining animals inGroup IV had died. The lesions in the surviving fifth animal wereresolving at day 14 post-challenge.

[0067] At 24 hours post-challenge, one of six animals in Group V haddied and the remaining five had developed ulcerated abdominal lesions.By 48 hours post-challenge, three of the five remaining animals in GroupV had died. One additional animal died by day 5 post-challenge. Thelesions in the surviving sixth animal were resolving at day 14post-challenge.

[0068] In contrast, all six of the animals in Group VI challenged withFLL32 survived the 14-day post-challenge observation period. None of theanimals in Group VI had any observable negative effects from thechallenge.

[0069] The results of these challenges were analyzed using Fisher'sExact Test. The analysis found no difference in the virulence betweenW83 and FLL33 (p=0.727). However, the FLL32 strain had a statisticaldifference in virulence when compared to FLL33 (p=0.008) and W83(p=0.015).

[0070] As can be appreciated from this Example, the inactivation of therecA gene in P. gingivalis FLL33 did not significantly affect thevirulence of P. gingivalis. However, the mutation in the FLL32 strainsignificantly affected the virulence of P. gingivalis.

EXAMPLE II Demonstration of the Protective Effect of Immunization withFLL32 Against Subsequent Challenge with Wild-Type W83 P. Gingivalis

[0071] The protective effect of immunization of a mammal with FLL32against subsequent challenge with wild-type W83 P. gingivalis wasdemonstrated as follows. Sixteen female Balb/c mice (8-10 weeks old,Harlan Sprague Dawley Inc., Indianapolis Ind.) were subcutaneouslyimmunized once per week for 3 weeks with 1×10¹⁰ bacteria of the mutantstrain FLL32, a dosage of 5×10⁵ bacteria per kg of body weight. Tenadditional female Balb/c mice (8-10 weeks old, Harlan Sprague DawleyInc., Indianapolis Ind.) were subcutaneously immunized once per week for3 weeks with sterile phosphate-buffered saline (PBS) as a control. Allof the animals immunized with FLL32 and five of the ten animalsimmunized with PBS were then challenged 2 weeks after the finalimmunization by subcutaneous injection of a P. gingivalis W83 wild-typesuspension containing 1×10¹⁰ cells, a dosage of 5×10⁵ bacteria per kg ofbody weight. The remaining five animals immunized with PBS werechallenged 2 weeks after the final immunization by subcutaneousinjection of PBS as a control.

[0072] By 24 hours post-challenge, one of the five control animalsimmunized with PBS and challenged with P. gingivalis W83 died and theother four animals had developed spreading infections with secondarysite abdominal skin ulcerations and, in some, primary site ulcerationsaround the base of the tail. All of these mice exhibited severe cachexiawith ruffled hair, hunched bodies and weight loss; and all of these fivecontrol animals died by four days post-challenge.

[0073] In contrast, eight of sixteen animals immunized with P.gingivalis FLL32 and challenged with P. gingivalis W83 displayed onlyminor secondary skin site abdominal infections by 24 hourspost-challenge but all recovered and were alive at the end of the testperiod. Of the remaining eight animals immunized with P. gingivalisFLL32 and challenged with P. gingivalis W83, five had severe cachexiaand died by three days post-challenge, two had moderate cachexia anddeveloped secondary ulcerating abdominal lesions which began to heal atday 5 post-challenge and were alive at the end of the fourteen dayexperiment period, and the last animal developed a secondary lesionwhich healed but then developed an additional secondary lesion and diedat day 7 post-challenge.

[0074] All of the five animals immunized with PBS and challenged withPBS appeared normal throughout the fourteen-day experiment period.

[0075] The results of these challenges were analyzed using Fisher'sExact Test. The analysis found that immunization with the FLL32 strainprotected the animals from a wild-type challenge (p=0.148), while thoseanimals that were immunized with sterile phosphate-buffered saline werenot protected (p=0.023).

[0076] At the end of the fourteen-day experiment period, the tensurviving animals from the group originally immunized with FLL32 andthen challenged with W83, and the five animals immunized with PBS andchallenged with PBS were sacrificed and their sera were isolated toascertain the presence of anti-FLL32 antibodies. A 1:1000 dilution ofthe sera was tested by Western blot analysis for cross-reactivity towhole cell lysates of P. gingivalis W83, FLL32 and FLL33.

[0077] Animals immunized with FLL32 were positive for antibodies to eachof the P. gingivalis whole cell lysates (data not shown). Immunoreactivebands with molecular mass of 96, 82, 74, 55.2, 49.6, 38, 37, and 35 kDawere observed in the Western blot analyses of each of the whole celllysates of FLL32, FLL33 and W83. Immunoreactive bands with molecularmass of 44 and 40 kDa were present in the Western blot analyses of eachof the whole cell lysates of FLL33 and W83 but were absent from theWestern blot analysis of the whole cell lysates of FLL32. Further,immunoreactive bands with molecular mass of 185, 170, 125, 71, 68, 63and 47 kDa were present in the Western blot analyses of whole celllysates of FLL32 but were absent in the Western blot analysis of each ofthe whole cell lysates of FLL33 and the W83 strain.

[0078] In contrast, sera from animals immunized with PBS and challengedwith PBS were negative for antibodies to each of the P. gingivalis wholecell lysates.

EXAMPLE III Method of Decreasing the Growth Rate or Reproduction Rate ofPorphyromonas Gingivalis in a Mammal

[0079] According to one embodiment of the present invention, there isprovided a method of decreasing the growth rate or reproduction rate ofPorphyromonas gingivalis in a mammal, such as a human. The methodcomprises the step of administering to the mammal at least one dose of anon-virulent, recA⁻ mutant of Porphyromonas gingivalis, such as FLL32.The dose can be administered, for example, by subcutaneous,intramuscular or intravenous injection. In a preferred embodiment, thedosage is between about 1×10³ and 1×10⁷ bacteria per kg of body weight.In a particularly preferred embodiment, the dosage is between about1×10⁵ and 1×10⁶ bacteria per kg of body weight.

[0080] Among the uses of decreasing the growth rate or reproduction rateof Porphyromonas gingivalis in a mammal, such as a human, is theprevention or treatment of periodontitis, or other diseases orconditions caused in whole or in part by Porphyromonas gingivalis, suchas aspiration pneumonia and necrotizing pneumonia, abscesses in brain,genitourinary tract and lung, and mediastinitis.

[0081] Although the present invention has been discussed in considerabledetail with reference to certain preferred embodiments, otherembodiments are possible. Therefore, the spirit and scope of theappended claims should not be limited to the description of preferredembodiments contained herein.

I claim:
 1. A non-virulent, recA defective mutant of Porphyromonasgingivalis.
 2. A pharmaceutical preparation comprising the mutant ofPorphyromonas gingivalis according to claim
 1. 3. A method of decreasingthe growth rate or reproduction rate of wild-type Porphyromonasgingivalis in a mammal, the method comprising administering to themammal at least one dose of the mutant of Porphyromonas gingivalisaccording to claim
 1. 4. The method of claim 3, wherein the mammal is ahuman.
 5. The method of claim 3, wherein the administration comprisesinjecting the mammal with the at least one dose of the non-virulent,recA defective mutant of Porphyromonas gingivalis via a route selectedfrom the group consisting of a subcutaneous route, an intravenous routeand an intramuscular route.
 6. The method of claim 3, wherein the doseadministered is between about 1×10³ and 1×10⁷ the mutant ofPorphyromonas gingivalis per kg of body weight of the mammal.
 7. Amethod of decreasing the growth rate or reproduction rate ofPorphyromonas gingivalis in a mammal, the method comprising the step ofadministering to the mammal at least one dose of a non-virulent, recAdefective mutant of Porphyromonas gingivalis.
 8. The method of claim 7,wherein the mammal is a human accession number 202109 to the mammal. 9.The method of claim 7, wherein the step of administering comprisesinjecting the mammal with the at least one dose of a non-virulent, recAdefective mutant of Porphyromonas gingivalis via a route selected fromthe group consisting of a subcutaneous route, an intravenous route andan intramuscular route.
 10. The method of claim 7, wherein the step ofadministering comprises injecting the [mutant] mammal with the at leastone dose of a non-virulent, recA defective mutant of Porphyromonasgingivalis, wherein the dose is between about a 1×10³ and 1×10⁷ bacteriaper kg of body weight.